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RecA acts intrans

to allow replication ofdamaged DNA by DNA polymerase VKatharina Schlacher1, Michael M. Cox2, Roger Woodgate3 & Myron F. Goodman1

The DNA polymerase V (pol V) and RecA proteins are essential components of a mutagenic translesion synthesis

pathway in Escherichia coli designed to cope with DNA damage. Previously, it has been assumed that RecA binds to the

DNA template strand being copied. Here we show, however, that pol-V-catalysed translesion synthesis, in the presence

or absence of the b-processivity-clamp, occurs only when RecA nucleoprotein filaments assemble or RecA protomers

bind on separate single-stranded (ss)DNA molecules in trans. A 30

-proximal RecA filament end on trans DNA is essential

for stimulation; however, synthesis is strengthened by further pol VRecA interactions occurring elsewhere along a trans

nucleoprotein filament. We suggest that trans-stimulation of pol V by RecA bound to ssDNA reflects a distinctive

regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis on a damaged template

strand obstructing translesion DNA synthesis despite the absolute requirement of RecA for SOS mutagenesis.

In 1974, Miroslav Radman proposed that E. coli possessed aninducible response to protect cells from the deleterious consequencesof DNA damage1. The hypothesis was elaborated on shortly there-after by Evelyn Witkin2, and the SOS response (as it has come to beknown) has been well characterized in the ensuing three decades andis now recognized to involve the upregulation of over 40 proteinsengaged in DNA repair, replication and recombination under thecontrol of the LexA repressor3. It has tacitly been assumed thatuncoupling of leading and lagging strand replication at blockinglesions results in unwound regions of ssDNA ahead of the stalledreplication fork, which serve as a platform for RecA nucleoproteinfilament formation4. If SOS-induced non-mutagenic repair processesare insufficient to reactivate cellular DNA replication, a mutagenicphase of SOS is initiated, facilitated largely by the translesionpolymerase, pol V (also known as the UmuD

0

2C complex)4. Trans-

lesion DNA synthesis (TLS) is generally successful in restarting DNAreplication, and is accompanied by a large (,100-fold) increase inmutations targeted principally at sites of DNA damage3.

Both in vitro58 and in vivo911, pol V activity is almost entirelydependent on the RecA protein. A RecA filament formed ondamaged DNA located cis to pol V has thus been the focus ofprevious studies on the molecular mechanisms of pol-V-dependentTLS8,12,13. Indeed, the role of RecA in TLS has progressed throughseveral iterations, from simply positioning UmuD

0

2C at the template

or primer14,15

, to a moving interaction that changes as the RecAfilament is displaced by pol V12, and, most recently, as an integralcomponent of pol V16 (Supplementary Fig. 1). However, one crucialfactor was overlooked in all of these studiesthe possibility thatRecA might act in trans, rather than in cis, to stimulate pol V.

Transactivation of pol V by RecA bound to ssDNA

Linear and closed-circular primer/template DNA contains eitherexcess primer or template strands. The use of hairpin DNA insteadof primer/template DNA ensures the absence of unannealed ssDNA.Pol V alone catalyses weak primer elongation on hairpin DNAcontaining a 3-nucleotide-long template overhang (Fig. 1a, lane 1;

Supplementary Fig. 2). RecA in the presence of ATP does notstimulate pol V (Fig. 1a, lane 2). However, the addition of a non-homologous ssDNA (trans 80-mer) activates RecA for robust primerextension by pol V (Fig. 1a, lane 3). These data demonstrate thatstrong stimulation of pol V occurs when RecA is activated by a transDNA moleculethat is, one that is not copied by pol V. (Supportivecontrol data are provided in Supplementary Fig. 3.)

We examined whether transactivation is required for pol Vactivitywhen the b-processivity-clamp is included in the reaction (Fig. 1b).To ensure efficient b-clamp loading by the g-clamp-loading com-plex, a hairpin with a 12-nucleotide overhang was used, and a biotinstreptavidin barrier was attached to prevent theb-clamp from slidingoff. Processivity of another SOS DNA polymerase, polIV, is enhanced(Fig. 1b, lane 5), verifying that the clamp has been loaded properly.However, polV is barelyactive in thepresenceof theb-clamp (Fig.1b,lane 1). RecA inhibits pol-IV-mediated synthesis in the absence orpresence of the b-clamp (Fig. 1b, lanes 6 and 7, respectively),indicating that RecA is able to interact with the hairpin DNA.Nonetheless, RecA is incapable of stimulating pol V (Fig. 1b,lane 2), unless trans ssDNA is also present (Fig. 1b, lane 3).

Pol V typically copies past DNA lesionsfor example, abasic sites(X in Fig. 1ce). However, it is only able to perform TLS when notonly RecA, but also ssDNA in trans, is present in the reaction (Fig. 1c,lanes 3 and 5). TLS is measured as the fraction of primers extended

past the lesion and is enhanced by approximately threefold from 7%to 22% in the presence of the b-clamp (Fig. 1c, lanes 3 and 5). Theenhancement of lesion bypass requires the presence of streptavidin-bound DNA to prevent dissociation of the b-clamp (Fig. 1c, lane 6).These data indicate that transactivation by RecA is necessary for bothnormal and translesion synthesis in the presence or absence of the b-clamp.

It has been a mechanistic question whether or not bona fide RecAfilaments were required for pol V TLS8,13 or whether smaller RecADNA complexes could suffice16,17. We extended the template over-hang to 50 nucleotides to determine whether transactivation is stillrequired when RecA filaments areformed in cis (Fig. 1d). As observed

ARTICLES

1Department of Biological Sciences and Chemistry, University of Southern California, University Park, Los Angeles, California 90089-2910, USA. 2Department of Biochemistry,

University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. 3Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human

Development, National Institutes of Health, Bethesda, Maryland 20892-2725, USA.

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with the shorter overhangs, pol-V-catalysed primer elongation (Sup-plementary Fig. 4) and TLS (Fig. 1d) is essentially inactive exceptwhen RecA and ssDNA are provided in trans. We verified that hairpinDNA containing a 50-nucleotide overhang stimulates autocleavageof LexA, showing that RecA, which can act as a co-protease, is notexcluded from the cis DNA (Supplementary Fig. 4).

DNA lesions can occur in the leading strand, modelled by theprimer/template hairpin DNA in Fig. 1d. In vivo, however, pol V

could also act on damaged lagging strand gaps. To address thepossibility of differential activation requirements on gapped DNAsubstrate, we confirmed the requirement for transactivation ondouble hairpins with or without lesions (Supplementary Fig. 5).

Primed ssDNA circles have been used previously to characterizethe effects of pol-V-catalysed TLS7,8,18,19. To ensure that our obser-vations are not an anomaly related to the use of hairpin DNA, a 30-mer primer was annealed to a synthetic 240-mer circle in 1:1

stoichiometry. As observed for the hairpin substrates, pol V is barelyactive in the presence of RecA, the b-clamp or both (Fig. 1e, lanes14; Supplementary Fig. 6). However, robust synthesis and TLS areobserved when unprimed single-stranded circular DNA mimickingthe presence of excess unannealed circular template is provided intrans, with processivity enhanced by the b-clamp (Fig. 1e, lanes 5and 6; Supplementary Fig. 6). Thus, the addition of separatetransactivating ssDNA molecules seems to be a stringent requirement

for RecA-mediated stimulation of pol V synthesis on damaged andundamaged DNA templates (Fig. 1).In vivo data indicate that gaps persist and disappear much later

during the SOS response20, so the majority of RecAssDNA com-plexes in cells induced for SOS could occur within gapped DNA.Therefore, in comparison with transactivating single-stranded linearDNA, hairpin DNA and single-stranded circular DNA, we deter-mined that RecA bound to ssDNA gaps also transactivates pol V

Figure 1 | RecAssDNA transactivation of pol V on damaged and

undamaged DNA templates. a, Transactivation of pol-V-catalysedsynthesis on a hairpin DNA with a 3-nucleotide (nt.)-long templateoverhang (oh.) by a ssDNA (80-mer)RecA complex present in trans (lane3). b, RecADNA transactivation of pol V in the presence of theb/g-sliding-clamp and clamp-loading complex (left gel); control reactions with pol IV(5 nM) (right gel). St denotes the presence of streptavidinbiotin blocks.c, Transactivation of pol-V-catalysed TLS. X denotes the site of the DNA

lesion (an abasic site). d, Transactivation of pol V synthesis on a DNAhairpin containing a 50-nucleotide-long template overhang with a lesion, X.e, Transactivation of pol Von damaged (X) circular DNA by RecA bound totrans circular DNA. f, Sketchof transactivating DNA. In ae, thearrowheadsindicate the position of the full-length product; % PU is the percentage ofhairpin templates extended (primer utilization); transactivating DNAmolecules are at 160 nM, when present; and RecA is at 4 mM, when present.In ce, % TLS is the percentage of primers extended past the lesion, X.

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(Supplementary Fig. 7). The degree of transactivation is greater forlonger ssDNA regions. These data show that trans-stimulation of pol

Voccurswhen RecAbinds to linear ssDNA(Fig. 1af),or any formofgapped DNA (Supplementary Fig. 7).

Transactivation kinetics of pol V by RecA bound to ssDNA

To facilitate the characterization of the transactivation kinetics,reactions were performed using the slowly hydrolysable ATP-gS

(Fig. 2), which inhibits filament disassembly. Synthesis was measuredon 3-nucleotide-overhang hairpin DNA substrates to eliminateinhibition by the cis RecA filaments that can assemble only on longerssDNA overhangs (Supplementary Fig. 8). A linear relationship isobserved between the velocity of pol V synthesis and the concen-tration oftrans-DNARecA complex. The second-order dependenceof pol V activity on the trans-DNARecA complex is evidence of abimolecular transactivation reaction (Fig.2b). The specific activity of

pol V increases 30-fold in the presence of 5 nM trans ssDNA, which issubstoichiometric to the hairpin DNA (Fig. 2a, lane 2), comparedwith pol V in the absence of trans DNA (Fig. 2a, lane 1). A fourfoldexcess oftrans ssDNA (80 nM) over hairpin DNA (20 nM) resultsin a400-fold increase in the specific activity of pol V (Fig. 2a, lane 5). Aneightfold excess oftrans (160 nM) over hairpin (20 nM) DNA resultsin complete primer utilization within 20 min (Fig. 2, lane 6), andeven within 5 min (Supplementary Fig. 9).

Pol V transactivation requires a free 30

RecA filament end

The 30

-proximal end of the RecA nucleoprotein filament on transssDNA has a crucial stimulatory role (Fig. 3). A single-stranded 30-mer was biotinylated at the 5

0

end and attached to a streptavidin-coated magnetic bead (Supplementary Fig. 10). The trans-activatorbead strongly stimulates pol V activity (Fig. 3a, lane 2). When the

biotin tag is switched to the 3 0 end of the DNA, RecAwill nucleate onthe DNAbead complexes with the same 5

0

-to-30

directionality. Inthis case, however, thebead blocks accessto the3

0

-proximal end.Thisinverted filament is substantially less effective in stimulating pol V(Fig. 3a, lane 3), even though comparable amounts of RecA arebound to each of theDNAbeadcomplexes (Supplementary Fig. 11).

Figure 2 | Kinetics of pol V transactivation by RecAssDNA. a, Pol-V-catalysed primer utilization when copying a hairpin containing a3-nucleotide-long template overhang was measured in the presence of ATP-gS and RecA (2 mM) at varyingtrans ssDNA (36-mer) concentrations (innM).The specific activityof pol V ismeasuredin pmol DNA per mgpolVpermin. The arrowhead indicates the position of the full-length product. b,Primer extension velocity depends on trans DNA concentration. The kineticdata used to calculatethe reaction velocity is shown in SupplementaryFig. 9.

Figure 3 | A RecA filament end oriented 30

on trans DNA is required for

efficient stimulation of pol V. a, RecA protein (,4 mM) immobilized on aDNAbead complex (,1 mM of DNA molecules bound to the bead) withexposed 3

0

-proximal RecA ends causes strong transactivation of pol V. Afilament with exposed 5

0

ends shows weak transactivation. b, RecA (2 mM)bound to a hairpin DNA containing a 3

0

overhang stimulates pol V muchmorethanonewith a 5

0

overhang. c, RecX protein inhibits polV synthesisinthe presence of RecA (1 mM), trans 36-mer (80 nM) and ATP-gS (left panel),or RecA (4 mM), trans 80-mer (160nM) and ATP (right panel). d, Mutant

RecA1730 (4 mM) in the presence of ATP-gS and trans 80-mer (160 nM) isdeficient in transactivation. e, Mutant RecADC17/730(E38K) (,1 mM)immobilized on a 12-mer-DNAbead complex (,1 mM of DNA moleculesbound to the bead) causes robust transactivation. f, Wild-type (WT) andmutantRecA proteins (,4 mM) immobilized on a 30-nucleotide-DNAbeadcomplex stimulate pol V synthesis with significantly different efficiencies.Hairpin primer/template DNA contains a 3-nucleotide-long templateoverhang (see diagram in Fig. 2a). (See Supplementary Methods andSupplementary Fig. 10 for preparation of RecADNA bead complexes.)

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The requirement for an accessible 30

-proximal RecA filament endon ssDNA for pol V stimulation was confirmed by using hairpinoligonucleotides with 3

0

or 50

overhangs as the trans-activator DNA(Fig. 3b). Presumably, RecA filamentation on hairpins with a 5

0

overhang continues into the double-stranded regions, thereforeexcluding a 3

0

end on the ssDNA. The regulatory protein21 RecXblocks RecA filament extension by binding 3

0

to filament ends22.RecX also inactivates RecA transactivation of pol V, most likely via a

steric block, even when RecA is formed on the DNA with the slowlyhydrolysable ATP-gS before the addition of RecX (Fig. 3c, left panel).RecX is even more inhibitory in the presence of ATP (Fig. 3c, rightpanel). The RecA1730 mutant protein confers an SOS non-mutablephenotype10,23, and is unable to stimulate pol V activityin vitro16. Thesame holds true for the transactivation reactions (Fig. 3d). In theRecA1730 mutant, a phenylalanine replaces a serine at amino-acidresidue 117, a position that is located at the surface facing 3

0

on aRecA filament end15.

Hierarchy of pol V transactivation by RecA mutants

Mutant RecADC17/730(E38K)24 associates tightly with ssDNA16 andis able to transactivate pol V while attached to a 12-mer-DNAbeadcomplex (Fig. 3e, lane 2). RecA12-mer-DNA complexes do notprovide sufficient space for a complete helical turn of a RecA

filament. Notably, owing to the presence of the beads, these shortcomplexes cannot stack to form longer filaments. Thus, the datasupport the idea that no more than a small RecA protomer isnecessary for transactivation. Nevertheless, a longer trans ssDNAoligomer(30 nucleotides) increases RecA stimulatory activity (Fig.3f,left panel, lane 4).

The three mutant RecA proteins RecADC1724, RecA730(E38K)25

and RecADC17/730(E38K)16 all bind tighter to DNA than wild-typeRecA, and for that reason were believed to enhance RecA function.However, RecA binding properties are irrelevant in experiments withmagnetic beads because comparable amounts of RecA protein arestably bound to the DNAbead complex (Supplementary Fig. 11).The various RecA proteins show a hierarchy in pol V stimulation(Fig. 3f). Mutant RecADC17 is missing its carboxy-terminal lobe,

which otherwise occupies parts of the helical groove in a RecAfilament26. Therefore, this deletion may facilitate access to the groovefor proteins such as UmuD

0

, as has been modelled previously27.RecADC17 doubles the reaction velocity (Fig. 3f, right panel) andprimer utilization by pol V (Fig. 3f, lane 2) compared with wild-typeRecA. It is therefore tempting to speculate that the additionalstimulation seen in the presence of RecADC17 is attributable to afacilitated interaction between UmuD

0

and the helical groove of aRecA filament, as suggested in a previous study using electronmicroscopy27.

Discussion

Here we propose a model for the role of pol V in SOS DNA-damage-induced mutagenesis in which an interaction between pol V and thesurface of a RecA protomer or 3

0

-proximal RecA filament end is both

necessary and sufficient for lesion bypass (Fig. 4a, orangered circle).On the basis of in vivo studies with RecA mutants, Devoret and co-workers15,28,29 proposed that a 3

0

-proximal RecA filament end inter-acting with UmuD

0

C (later identified as pol V (UmuD0

2C)4) at a site

of DNA damage in cis was necessary for TLS. Conversely, the centralconclusion of the transactivation model is that interactions betweenpol V and RecA responsible for synthesis stimulation must occurwhile the proteins are bound to different molecules of DNA. Thiseffect was obscured in previous studies investigating pol-V-catalysedTLS from different groups7,8,13,18,19,30, including ours5,6,12,16,17, wheresome fraction of the ssDNAcould alwayshavebound RecA capable ofactivating pol V complexes on other primer/template molecules intrans.

The activity of pol V increases linearly with the concentration ofssDNA supplied in trans (Fig. 2b). The bimolecular nature of the

reaction represents strong kinetic evidence that stimulation of pol Vby RecA per se is dependent on a second trans DNA molecule boundto RecA. Complete hairpin DNA extension is observed on DNAsubstrates that preclude RecA inhibition in cis (Fig. 2a). The presenceor absence of the b-clamp does not alter the requirement that RecAbound to DNA in trans is required for pol-V-catalysed TLS (Fig. 1).

RecA nucleation on ssDNA can occur at any segment, and isfollowed by filament extension in the 3

0

direction31 while disassem-

bly, driven by ATP hydrolysis, occurs with the same directionality,thus creating new nucleation sites. Therefore, free 30

-proximal RecAfilament ends will continuously be generated on ssDNA, such as (forexample) circular DNA (Fig. 1e) or gapped DNA (SupplementaryFig. 7), and can serve as transactivation docks for pol V. Presumably,the longer the single-stranded region stretches, the more RecAfilaments are able to initiate, thereby increasing the number ofavailable 3

0

-proximal ends. Notably, a gapped circle containinglonger ssDNA (Supplementary Fig. 6) or an unprimed single-stranded 240-mer circle (Fig. 1e, lane 5) provided in trans increasesthe extent of pol V stimulation.

An unambiguous consequence of a stalling replisome or replica-tion fork collapse on an encounter with a DNA lesion is the creationof ssDNA regions. This is a key event for the initiation of the SOSresponse. Accurate repair mechanisms, such as recombinational

repair or excision repair, all of which involve or create ssDNA, tryto cope with the damage before inducing mutagenic pol V. However,gaps persist20 and disappear only very late during the SOS response.Coincidentally, pol V is also produced late in the SOS response,presumably as a last resort to facilitate cell survival.

We suggest that regulation of SOS mutagenesis requires elevatedlevels of ssDNA in trans for optimal pol Vactivation (Fig. 2). Simpleaccess to a stalled replication fork and high amounts of RecA areinsufficient for appreciable pol-V-catalysed synthesis. Mutagenesis isrestricted to times of extensive DNA damage: for example, whennumerous ssDNA gaps are present, thus increasing the chances for assDNA gap transactivator (Fig. 4b) to stimulate pol V on a separatereplication fork. Any segment on ssDNA, if sufficiently long, canserve as a RecA filament platform (Fig. 4b). Once the damage is

bypassed and extended, ssDNA disappears and pol V activitiesdecline. The requirement for RecA during pol-V-catalysed TLS in

Figure 4 | Models depicting RecADNA transactivation of pol-V-catalysed

translesion DNA synthesis. a, RecAssDNA(orange circles) is required forpol V (UmuD

0

2C) to copy undamaged and damaged DNA templates. Theessentialfeature of thetransactivationmodelis that a RecADNAcomplex isformed on ssDNA that is not being copied by pol V. If RecA were to bindinstead to the template strand, cis to pol V, then TLS would be blocked. b, A

variation on the model shows how transactivation of pol V might occur by aRecA filament formed in gapped DNA (blue). A damaged DNA base isshown as a distortion in the template strand (brown squiggle).

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vivo and in vitro seemed incompatible with the observation that RecAfilaments formed in cis on the template being copied can block DNAsynthesis12,16 (Supplementary Fig. 12). Activation of pol V by RecAssDNA in trans resolves this incompatibility and seems to constitute anew type of polymerase regulatory mechanism.

METHODSNucleotide incorporation on DNA primer templates. The polymerase activity

was examined by extending primer/template hairpin DNA32

P-labelled at the 50

end, or 32P-labelled primed circular DNA, as indicated. Substrate DNA waspreincubated with b-processivity-factor/g-clamp-loading complex (when pre-sent) and pol V before addition of RecA, trans DNA and dNTPs. Preincubationoftrans DNA with RecA is redundant, as RecA will be attracted to DNA that islonger and/or in molar excess over primer/template DNA. RecA concentrations,trans DNA length or concentrations, and the presence of either ATP or ATP-gSare indicated in the figures and figure legends. The sequences of template andtransactivating DNAs are provided in Supplementary Fig. 13. For DNA beads, abiotinylated oligomer was attached to a streptavidin-coated magnetic bead(Bangs Laboratories) following the manufacturers protocol. RecA was immobi-lizedon thebeads withATP-gS asdescribed inSupplementaryFig. 10, andaddedto the reaction at a concentration of,1mM in total DNA molecules. Unlessindicated otherwise, reactions were carried out for 20 min at 37 8C and thesynthesis products were separated on a 20% denaturing polyacrylamide gel. Thegel band intensities were measured by phosphorimaging with IMAGEQUANT

software (Molecular Dynamics) and primer utilization was computed from theintegrated gel band intensities of extended hairpin DNA or primer DNA. TLSwas calculated by dividing the number of hairpins extended past the abasic siteby the total number of extended hairpins. Transactivation kinetics curves(Supplementary Fig. 9) were analysed using Sigma Plot. For further details onthe methods used in this study, see Supplementary Methods.

Received 1 April; accepted 4 July 2006.

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements This work was supported by National Institutes of Healthgrants to M.F.G. and M.M.C., and funds from the NICHD/NIH IntramuralResearch Program to R.W.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Correspondence and requests for materials should be addressed to M.F.G.

(mgoodman@usc.edu).

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